The focus of the proposed research is the structure, function and catalytic mechanisms of three non-heme iron enzymes involved in oxidative or nitrosative stress protection in air-sensitive bacteria. All of the enzymes proposed for study reductively scavenge diatomic molecules that are toxic to air-sensitive bacteria at micromolar or lower levels. The enzyme, superoxide reductase (SOR), scavenges superoxide resulting from the bacteria's accidental one-electron reduction of oxygen. The enzyme, rubrerythrin (Rbr), scavenges hydrogen peroxide resulting from accidental two-electron reduction of oxygen. The flavo-diiron enzyme, FprA, scavenges nitric oxide generated both internally and externally, e.g., from symbiotic bacteria or the intestinal epithelium. Hundreds of bacterial species occupy the human colon and oral cavity, and the vast majority are air-sensitive. These enzymes also conceivably protect against the oxidative and nitrosative burst of macrophages, which is the mammalian host's initial response to infection, but very few studies of these enzymes from pathogens have been conducted. The enzymes listed above from two human periodontal pathogens and from a food-borne human gastric pathogen are among those that will be investigated. Since the catalytic reactions of these enzymes occur on microsecond to millisecond time scales at room temperature, experiments are proposed to either rapidly mix and trap the reaction intermediates or to slow down the reactions using cryogenic temperatures. Both approaches should then allow structural and electronic characterizations of these intermediates using various spectroscopies and X-ray crystallography. The goals are to obtain "snapshots" of the reaction progress and to correlate these reaction mechanisms with the protective roles of these enzymes in vivo. Many of the bacteria that contain SOR, Rbr and FprA have become resistant to the available antibiotics. Since homologues of these enzymes have not been found in humans or animals, inhibitors of these enzymes could conceivably be developed into new classes of antibiotics. The detailed understanding of the catalytic mechanisms of these enzymes and their protective roles resulting from the proposed studies will be essential for the intelligent design of such antibiotics.